Preliminary Study On The Application Of DNA Fingerprinting And Phenotypic Feature Inference Techniques Based On Polymorphic Genetic Markers In Individual Identification

Objective: To explore the application value of DNA fingerprinting and phenotypic feature inference technology based on polymorphic molecular genetic marker in the identification of individual living things by verifying the polymorphic genetic marker compound detection system Si Fa STR 23 plex and the use of human iris color and hair color related genetic marker to form a phenotypic feature inference system.method:1.Study subjects:(1)A population of 302 unrelated individuals of the Han ethnic group in Fujian who visited Fujian Provincial Hospital from September 2021 to December 2021 was selected as the study subjects(205 males and 97 females).2m L of venous whole blood was extracted from each study subject,anticoagulated with EDTA-K2,and stored at-20℃.Extract sample DNA using the Tiangen Blood Genome DNA Extraction Kit.(2)Among the above study subjects,50 randomly selected cases were compared for genotype consistency at the same STR locus in different detection systems.(3)260 individuals aged between 18 and 35 were selected as representative molecular research subjects from the group study,with 175 males and 85 females.Collect images of its hair color and iris color,and manually interpret and classify them according to the color classification standards in the literature.2.DNA fingerprinting research-verification of the polymorphic genetic marker complex detection system(1)In this study,we used Si Fa STR 23 plex,a polymorphic genetic marker compound detection system,to detect 23 genetic marker(21 autosomal STR loci,1 Y-Indel locus,and 1 sex locus Amelogenin)combined with four fluorescent dyes.DNA template and genetic marker primer are added into the PCR reaction system,and the fluorescence signal of genetic marker is detected by multiplex amplification,capillary electrophoresis and fluorescence signal detection.The obtained fluorescence signal is combined with allelic standard and molecular weight standard labeled with orange fluorescent dye to interpret the genotype of genetic marker.The sensitivity of the detection system and the accuracy of genetic marker genotyping results were verified by using DNA reference material(No.9948).(2)The allelic typing results of 21 autosomal STR loci were input into Power Stats v12 analysis software to calculate population genetics parameters such as heterozygosity,polymorphism information content,individual recognition ability,etc.The haplotype frequency of Y-Indel locus was counted by Excel software,The detection efficiency of DNA fingerprinting consisting of 21 autosomal STR loci and 1 Y-Indel locus in individual identification of Fujian Han population was evaluated by population genetics parameters.(3)SPSS v23 software was used to carry out Kappa test on the consistency of the typing results of the same 20 autosomal STR loci in the Si Fa STR 23 plex composite detection system and the foreign composite detection system Power Plex 21 detection system(Promega Company of the United States).2.Phenotypic feature molecular research-constructing a characterization molecular detection system related to human iris color and hair color(1)Search for data,synthesize single nucleotide polymorphism(SNP)site primers,and construct a composite amplification system of 24 characteristic SNP sites related to iris color and hair color.(2)The genotyping data of 24 characterization molecular SNP sites of 260 subjects were obtained by PCR multiplex amplification and capillary electrophoresis typing,and the chi square test of the minimum allele frequency frequency of 24 characterization molecular SNP sites in different regions was performed by SPSS v23 software to compare their differences;Upload SNP typing results to the HIris Plex phenotypic characteristics online inference model at the University of Erasmus in the Netherlands(https://HIris Plex.erasmusmc.nl/)Perform online inference on the hair color and iris color of the research object.Compare the online inference model results with the manual interpretation classification results,use SPSS v23 software to draw the ROC curves of each pigment representation inference result and calculate the AUC value,and evaluate the inference efficiency of the system for the iris color and hair color representation of the Han population in Fujian region.Results: 1.Study on DNA fingerprint(1)The DNA concentration of Si Fa STR 23 plex,a polymorphic genetic marker complex detection system,is 5ng/ μL-62.5pg/ μL Within the range,all 21 autosomal STR loci and 1Y-Indel locus haplotype can be obtained.Two cases of abnormal genotype were found in the study,one of which was the D1S1656 locus with a triple band allele of 14/15/17.3;Another case of amelogenin deletion at the gender locus was found for AMEL-X genotyping.(2)To establish a DNA genetic information database based on 21 autosomal STR loci and1 Y-Index locus from 302 unrelated individuals of Han nationality in Fujian Province.The polymorphism information content of 21 autosomal STR loci is 0.58-0.91,with an average individual recognition rate of 0.928 and an average heterozygosity of 0.783.The cumulative individual recognition ability of the detection system is greater than0.9999999999,and the cumulative non paternal exclusion rate is greater than 0.999999992.The allele data of all STR loci are consistent with Hardy Weinberg equilibrium(p>O.05).(3)50 randomly selected research subjects were tested using two systems,and the consistency of the typing results of 20 identical autosomal STR loci was determined by Kappa test.The Kappa value was equal to 1,and the results were completely consistent.2.Phenotypic characteristic molecular research(1)A composite detection system of 24 characterization molecular SNP sites related to iris color and hair color was constructed.The SNP genotyping results of 260 unrelated individuals from the Han nationality in Fujian Province showed 12 polymorphisms,including rs885479,rs1805005,rs2228479,rs28777,rs4959270,rs12203592,rs1042602,rs2402130,rs12913832,rs2378249,rs12896399,and rs683.Compared with the European population,the minimum allele frequency of the other 24 SNP loci in the Han population in Fujian Province was significantly different by chi square test(p<0.05),except N29 ins A,rs Y152 OCH,and rs12896399.(2)The typing results of 24 characteristic molecular SNP sites were analyzed using the HIris Plex online inference model.The iris color of 260 study subjects was manually interpreted as 259 brown and 1 intermediate color,all of which were inferred as brown by the online model;The hair colors of 260 study subjects were manually interpreted as 248 black and 12 brown,all of which were inferred to be black hair through online models.The HIris Plex online inference model has a 100% accuracy rate and AUC value of 0.947 for brown eyes and 0.817 for black hair in the Han population of Fujian region.Conclusion:(1)Si Fa STR 23 plex,a polymorphic genetic marker complex detection system,has the characteristics of high sensitivity,simple operation and easy interpretation of typing results.The DNA fingerprint map established based on the 21 autosomal STR loci,1 Y-Indel locus,and Amelogenin gender locus of this system has the advantages of high cumulative individual recognition ability and rich polymorphism information,which can meet the requirements of individual identification in forensic material evidence identification.It has certain application value in the genetic identification of the Han population in Fujian region.(2)The constructed SNP site detection system for 24 characterization molecules related to iris color and hair color has stable typing results and is easy to analyze.The HIris Plex online inference model has high accuracy in inferring the phenotypic characteristics of iris color and hair color in the Han population in Fujian Province.Due to population and ethnic differences in the genotyping results of characterization molecular SNP loci,further research should be conducted on the phenotypic characteristics of hair color and iris color,especially in the study of hair color related genes,and more characterization molecular SNP loci should be introduced to improve the accuracy of the inference results.(3)In conclusion,DNA fingerprinting and phenotypic feature inference technology composed of polymorphic genetic marker can have good application prospects in individual identification of Han population in Fujian.It can provide powerful technical means and effective identification methods for individual identification of organisms in terms of genetic polymorphism and the association between genes and phenotypic features.